Objective: To examine the potential role of the L-arginine:nitric oxide pathway in hCG-induced ovulation in the rabbit.Design: Randomized, controlled animal study.Setting: University research laboratory.Intervention(s): Nitric oxide synthase, the enzyme that produces nitric oxide (NO), was immunohistochemically localized in the ovary. N-G-nitro-L-arginine methyl ester (L-NAME), an analogue of L-arginine, which inhibits the enzyme NO synthase, and the inactive D-enantiomer were administered in vivo and/or in vitro via an isolated, perfused ovary preparation during the periovulatory period.Main Outcome Measure(s): Rate of follicular rupture (ovulatory efficiency).Result(s): Immunohistochemical staining for NO synthase was localized specifically to the granulosa cell layer of the follicle and the endothelium and adventitia of ovarian blood vessels. In vivo administration of L-NAME significantly reduced the percentage of large follicles that ovulated in response to hCG (treated 24.6%, control 68.1%). Similarly, exposure of the in vitro-perfused ovary to L-NAME significantly reduced follicular rupture (treated 32.8%, control 64.2%). In contrast, addition of an equimolar concentration of D-NAME to the perfusion medium had no significant effect on the rate of ovulation (treated 83.3%, control 61.3%).Conclusion(s): The stereospecific inhibition of follicular rupture by the arginine analogue suggests that NO production by the ovary is an important feature of the normal physiologic processes of the periovulatory period.